CA3205490A1

CA3205490A1 - Use of pharmaceutical composition for treating lung cancer

Info

Publication number: CA3205490A1
Application number: CA3205490A
Authority: CA
Inventors: Chang-Hai TSAI; Mien-Chie Hung; Sheng-Chu Kuo; Pei-Chih LEE; Min-Tsang Hsieh; Shin-Hun Juang; Hui-Yi Lin
Original assignee: Individual
Current assignee: HUNG MIEN CHIE
Priority date: 2020-12-17
Filing date: 2021-12-14
Publication date: 2022-06-23
Also published as: TW202225136A; KR20230131861A; JP2023554532A; CN117120438A; TWI798994B; WO2022127751A1; AU2021402788A1; AU2021402788A9; EP4265256A1; US20240000742A1; EP4265256A4

Abstract

A use of a pharmaceutical composition is provided. The pharmaceutical composition contains a diarylheptanoid compound or a pharmaceutically acceptable salt thereof. The diarylheptanoid compound has a structure shown in formula (I). Symbols in formula (I) are as defined in the description, and the pharmaceutical composition can inhibit the growth of lung cancer cells. Therefore, the pharmaceutical composition can be used for preparing a drug for treating lung cancer.

Description

USE OF PHARMACEUTICAL COMPOSITION FOR TREATING LUNG CANCER
Technical Field The present disclosure relates to a use of a pharmaceutical composition. More particularly, the present disclosure relates to a use of a pharmaceutical composition including a diarylheptanoid compound or a pharmaceutically acceptable salt thereof for treating lung cancer.
Description of Related Art Cancer, also known as malignant tumor, is an abnormal proliferation of cells, and these proliferating cells may invade other parts of the body, which is a disease caused by abnormal control of cell division and proliferation mechanisms. There is an increasing trend in the number of people suffering from cancer worldwide, and about 20% of the cancer population in the world is lung cancer patients. The 5-year survival rate of lung cancer patients after treatment is still as low as about 15%, which has been the cancer with the highest death rate in the world for many years.
According to different biological characteristics, treatment and prognosis, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer (NSCLC).
About 85-90% of lung cancers are NSCLC, of which lung adenocarcinoma is the most common type of lung cancer in women and non-smoking patients. Treatment for lung cancer often depends on the age of patient, past medical history, current health status, type of cancer cells, and stage of the disease.
Generally speaking, small cell lung cancer has the characteristics of rapid division Date Regue/Date Received 2023-06-16 and proliferation, and metastases may occur in a short period of time, so that systemic chemotherapy or radiation therapy is the main treatment. The growth of NSCLC
is slower, and the occurrence of metastasis is also slower, so that the principle of treatment depends on the clinical stage of the disease. The radical treatment of early stage (stage I, II) NSCLC is still based on complete resection of the tumor by surgery. The treatment principle is mainly chemical drug therapy or chemical drug combined with radiation therapy for the locally extended stage (stage III) including patients with malignant pericardium or hydropleural effusion and distant metastases (stage IV) or patients whose physical condition cannot be surgically removed.
However, it is a thorny problem in the treatment of metastatic or advanced NSCLC that has undergone chemotherapy and relapsed. Current clinical studies have confirmed that epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs) can be used as the second-line treatment after the first-line chemotherapy fails. But about 40-80% of NSCLC patients have the EGFR gene mutation, which overexpresses the epidermal growth factor receptor, leading to rapid growth, metastasis and drug resistance of cancer. Almost all patients with the EGFR
gene mutation relapse within two years after clinical treatment with EGFR-TKIs, and no effective drugs are available after relapse so far.
SUMMARY
In view of this, one of the objectives of the present disclosure is to provide a use of a pharmaceutical composition, which can be used to manufacture a drug for treating a lung cancer. The pharmaceutical composition includes a diarylheptanoid

2 Date Regue/Date Received 2023-06-16 compound or a pharmaceutically acceptable salt thereof, which can inhibit the growth of the epidermal growth factor receptor-tyrosine kinase inhibitors resistant non-small cell lung cancer cells. Therefore, the pharmaceutical composition can be used alone or in combination with clinically used epidermal growth factor receptor-tyrosine kinase inhibitors to treat a EGFR gene mutation with epidermal growth factor receptor-tyrosine kinase inhibition drug-resistant lung cancer.
According to one aspect of the present disclosure is to provide a use of a pharmaceutical composition, the pharmaceutical composition is used to manufacture a drug for treating a lung cancer, wherein the pharmaceutical composition includes a diarylheptanoid compound or a pharmaceutically acceptable salt thereof, the diarylheptanoid compound has a structure represented by Formula (I):

Ra R ' a RC Rc Rb Rbi Re Re Formula (I), wherein Ra, Rb, Ra' and Rh' are independently H, C1-C2 alkyl, C1-C3 alkoxy, OH, or -0C(=0)Rd, wherein Rd is C1-C3 alkyl or C1-C3 alkanol; Rc is H, C1-C2 alkyl, C3-C6 unsaturated alkyl or C7-C12 arylalkyl with double or triple bonds; and Re and Re' are independently H, C1-C6 alkyl or C1-C6 alkoxy.
According to the use of the pharmaceutical composition, wherein at least one of Ra, Rh, Ra', and Rh' can be -0C(=0)Rd, wherein Rd is C1-C3 alkyl or C1-C3 alkanol.

3 Date Recue/Date Received 2023-06-16 According to the use of the pharmaceutical composition, wherein the diarylheptanoid compound can be interconvertible between keto and enol forms, when Rc is H.
According to the use of the pharmaceutical composition, wherein the diarylheptanoid compound can be selected from Compound 1, Compound 21a, Compound 21b, Compound 22a, Compound 22b, Compound 23a, Compound 23b, Compound 24a, Compound 24b, Compound 25, Compound 26, Compound 27, Compound 31 and Compound 33 having a structure represented by Formula (II):

nn R3 rs.3 Formula (II);
wherein Ri, Ri' are OCH3 respectively, R2, R2' are H respectively, R3, R3' are H
respectively in Compound 1; Ri, Ri' are OCH3 respectively, R2, R2' are 0R4 respectively, R3, R3' are H respectively in Compound 21a; Ri, Ri' are OCH3 respectively, R2 is OH, R2' is 0R4, R3, R3' are H respectively in Compound 21b; Ri, Ri' are OCH3 respectively, R2, R2' are 0R5 respectively, R3, R3' are H
respectively in Compound 22a; Ri, Ri' are OCH3 respectively, R2 is OH, R2' is 0R5, R3, R3' are H
respectively in Compound 22b; Ri, Ri' are H respectively, R2, R2' are 0R5 respectively, R3, R3' are H respectively in Compound 23a; Ri, Ri' are H
respectively, R2 is 0R5, R2' is OH, R3, R3' are H respectively in Compound 23b; Ri, Ri' are

4 Date Recue/Date Received 2023-06-16 respectively, R2, R2' are OCH3 respectively, R3, R3' are H respectively in Compound 24a; Ri is 0R4, Ri' is OH, R2, R2' are OCH3 respectively, R3, R3' are H
respectively in Compound 24b; Ri is 0R5, Ri' is OH, R2, R2' are OCH3 respectively, R3, R3' are H
respectively in Compound 25; Ri, Ri' are 0C2H5 respectively, R2, R2' are 0R4 .. respectively, R3, R3' are H respectively in Compound 26; Ri, Ri' are 0C2H5 respectively, R2, R2' are 0R5 respectively, R3, R3' are H respectively in Compound 27;
Ri, Ri' are C2H5 respectively, R2, R2' are 0R5 respectively, R3, R3' are C2H5 respectively in Compound 31; and Ri, Ri' are OCH3 respectively, R2 is OCH3, R2' is 0R4, R3, R3' are H respectively in Compound 33; wherein R4 is a structure represented by Formula (i), and R5 is a structure represented by Formula (ii):

OH OH
Formula (i); Formula (ii).
According to the use of the pharmaceutical composition, wherein the diarylheptanoid compound is selected from Compound 35a, Compound 35c, Compound 35d, Compound 35e, Compound 36 and Compound 37 having a structure represented by Formula (III):

R6 R6' '=,, Formula (III);

5 Date Recue/Date Received 2023-06-16 wherein R6, Re are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is CH3 in Compound 35a; R6, R6' are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is benzyl in Compound 35c; R6, Re are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is propargyl in Compound 35d; R6, Re are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is allyl in Compound 35e; R6, Re are H respectively, R7, R7' are 0R4 respectively, R8 is CH3 in Compound 36; and R6, Re are 0R4 respectively, R7, R7' are OCH3 respectively, R8 is CH3 in Compound 37; wherein R4 is a structure represented by Formula (i):

'''jts's4:0H
OH
Formula (i).
According to the use of the pharmaceutical composition, wherein the diarylheptanoid compound can be selected from:
. 0 Ai mectsoõ J ( .H Ofis 0 '-1 Mg __ He>510 IP 7\ ' W-1-le'"irk0 ---HO ii HO
"'OH , ''.
He>ft0L
11 'EOH Ho=-=>)., 1-cii0H
HO OH HO
,and o o 0 110">fk0 rigki 'N, ..--- OH
IHO Rip 40 ' If'OH
i-t3c0 OCH3 .

6 Date Recue/Date Received 2023-06-16 According to the use of the pharmaceutical composition, wherein the pharmaceutical composition can further include epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Preferably, the EGFR-TKIs can be osimertinib, gefitinib, erlotinib or afatinib.
According to the use of the pharmaceutical composition, wherein the lung cancer can be a non-small cell lung cancer (NSCLC).
According to the use of the pharmaceutical composition, wherein the lung cancer can be resistant to epidermal growth factor receptor-tyrosine kinase inhibitors.
The above summary is intended to provide a simplified summary of the disclosure to provide the reader with a basic understanding of the disclosure.
The summary is not an exhaustive overview of the disclosure, and it is not intended to identify key/critical elements of embodiments of the present disclosure or to delineate the scope of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
The present disclosure can be more fully understood by reading the following detailed description of the embodiment, with reference made to the accompanying drawings as follows:
Fig. 1A shows the analysis result of the inhibition of H1650 cell growth by the diarylheptanoid compounds of the present disclosure;
Fig. 1B, Fig. 1C and Fig. 1D show the analysis results of the inhibition of the growth of the epidermal growth factor receptor-tyrosine kinase inhibitors resistant

7 Date Regue/Date Received 2023-06-16 non-small cell lung cancer (NSCLC) cells by the diarylheptanoid compounds of the present disclosure;
Fig. 2A shows the analysis result of the inhibition of tumor growth in the GR6 tumor mice by treating Compound 35d alone;
Fig. 2B shows the analysis result of the inhibition of tumor growth in the GR8 tumor mice by treating Compound 35d alone;
Fig. 2C shows the analysis result of the inhibition of tumor growth in the tumor mice by treating Compound 35d alone;
Fig. 2D shows the statistical chart of body weight changes in the tumor mice by io treating Compound 35d alone;
Fig. 3A shows the analysis result of the inhibition of tumor re-progression in the GR6 tumor mice by treating a combined treatment of Compound 35d and osimertinib;
and Fig. 3B shows the statistical chart of body weight changes in the GR6 tumor mice by treating the combined treatment of Compound 35d and osimertinib.
DETAILED DESCRIPTION
The present disclosure provides a novel use of a pharmaceutical composition, and the pharmaceutical composition includes a diarylheptanoid compound or a pharmaceutically acceptable salt thereof, which can be used to manufacture a drug for treating a lung cancer. The diarylheptanoid compound of the present disclosure has a structure represented by Formula (I):

8 Date Regue/Date Received 2023-06-16 Re R ' a I
Rc Rc I , Re Re Formula (I), wherein Ra, Rb, Rai and RID' are independently H, C1-C2 alkyl, C1-C3 alkoxy, OH, or -0C(=0)Rd, wherein Rd is C1-C3 alkyl or C1-C3 alkanol; Rc is H, C1-C2 alkyl, .. C3-C6 unsaturated alkyl or C7-C12 arylalkyl with double or triple bonds;
and Re and Re' are independently H, C1-C6 alkyl or C1-C6 alkoxy.
At least one of Ra, Rb, Rai, and RID' of the diarylheptanoid compound of the present disclosure can be -0C(=0)Rd, and Rd is C1-C3 alkyl or C1-C3 alkanol. In addition, the diarylheptanoid compound can be interconvertible between keto and enol forms, when Rc is H.
The pharmaceutical composition of the present disclosure can further include epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs), which can be combined with the diarylheptanoid compound or a pharmaceutically acceptable salt thereof. The EGFR-TKIs can be osimertinib, gefitinib, erlotinib or afatinib.
The lung cancer treated by the pharmaceutical composition of the present disclosure can be non-small cell lung cancer (NSCLC). In addition, the lung cancer can be resistant to the EGFR-TKIs.
Unless otherwise noted, all terms, symbols or other scientific terms or terms used in the present disclosure have the meanings that are commonly understood by person having ordinary skill in the art. In some cases, terms with conventional meanings

9 Date Regue/Date Received 2023-06-16 are defined herein for clarity and/or immediate reference, and the definitions incorporated herein should be construed as not necessarily substantial different from the conventional meanings in the art. Many of the techniques and procedures described or referenced herein are well known and routinely used by those skilled in the art. Where appropriate, unless otherwise stated, procedures for the use of commercially available kits and reagents are generally performed according to instructions and/or parameters defined by the manufacturer.
Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms "a" and "an" mean one or more (that is at least one).
Furthermore, genera are recited as shorthand for a recitation of all members of the genus; for example, the recitation of C1-C3 alkyl is shorthand for a recitation of all C1-C3 alkyls including methyl, ethyl, propyl, and isomers thereof.
The diarylheptanoid compounds disclosed in the present disclosure and the pharmaceutically acceptable salt thereof can be verified by in vitro experiments, which can inhibit the growth of the EGFR-TKIs resistant NSCLC cells. It can further be verified by in vivo experiments that the compound disclosed in the specification and/or at least one pharmaceutically acceptable salt thereof can be administered to animals suffering from EGFR-TKIs resistant lung cancer (such as mouse model), and can obtain therapeutic effects. A positive result in one or more tests is sufficient to demonstrate the actual utility of the tested compound and/or salt, and an appropriate dosage range and administration route for animals (such as humans) can be determined based on test results.
Date Recue/Date Received 2023-06-16 Useful pharmaceutical dosage forms for administering the diarylheptanoid compound of the present disclosure and the pharmaceutically acceptable salt thereof include, but are not limited to, hard and soft gelatin capsules, tablets, parenteral injections and oral suspensions. The dosage administered can depend on factors including the age of the subject, the health and weight of the subject, the extent of the disease, the type of concomitant treatment (if any), the frequency of treatment and the nature of the desired effect. Usually the daily dose of active ingredient may vary, for example from 0.1 to 2000 mg per day. For example, 10-500 mg one or more times per day can be effective to achieve the desired results.
The same dosage form can generally be used when the diarylheptanoid compounds of the present disclosure and pharmaceutically acceptable salt thereof are administered stepwise or in combination with at least one other therapeutic agent.
When drugs are administered in a physical combination, the dosage form and route of administration should be selected based on the compatibility of the combined drugs.
Therefore, "co-administration" in the specification should be understood to include the concomitant or sequential administration of at least two agents, or as a fixed-dose combination of at least two active ingredients.
The diarylheptanoid compound and the pharmaceutically acceptable salt thereof in the specification can be used as the active ingredient alone, or administered in combination with at least one second active ingredient, the second active ingredient can be selected from, for example, other active ingredients known to be useful in the treatment of patients with NSCLC, in particular the EGFR-TKIs.

Date Regue/Date Received 2023-06-16 The following specific examples are used to further illustrate the present disclosure, in order to benefit the person having ordinary skill in the art, and can fully utilize and practice the present disclosure without excessive interpretation.
These examples should not be regarded as limiting the scope of the present disclosure, but is used to illustrate how to implement the materials and methods of the present disclosure.
1. Structure of the diarylheptanoid compound of the present disclosure The diarylheptanoid compound of the present disclosure uses curcumin (CCM) as a guiding compound to design a curcuminoid diarylheptanoid compound, which has a structure represented by Formula (I):

Re R ' a R R
c Rbi Re Re Formula (I), Please refer to the following Table 1, which shows Ra, Rb, Ra', Rc, Re and Re' of examples of the diarylheptanoid compound of the present disclosure -Compound 1, Compound 21a, Compound 21b, Compound 22a, Compound 22b, Compound 23a, Compound 23b, Compound 24a, Compound 24b, Compound 25, Compound 26, Compound 27, Compound 31, Compound 33, Compound 35a, Compound 35c, Compound 35d, Compound 35e, Compound 36 and Compound 37.

Date Recue/Date Received 2023-06-16 Table 1 Compound Ra Ra' Rb RID' Rc Re, Re' 21a OCH3 OCH3 ¨0-8 cH3 ¨0-8 cH3 H H
OH OH
O -OH
21b OCH3 OCH3 ¨0-8 cH3 OH H H
-OH

22a OCH3 OCH3 ¨0-g Ic2H5 ¨0-6 c2H5 H H
OH -OH
22b OCH3 OCH3 0 () c2H5OH H -- H
LOH
O [OH 0 [OH
23a H H ¨0-8 cH3 ¨0-8 cH3 H -- H
OH OH
O [OH
23b H H ¨0-8 8H3 OH H H
OH

24a ¨0-8 -OH -OH

24b ¨0-8 cH, -OH

LOH
O [OH 0 [OH
26 0C2H5 0C2H5 0-8 cH3 ¨0-8 cH3 H H
OH OH

27 0C2H5 0C2H5 0-8 c2H5 ¨0-8 c2H5 H H
OH OH

31 C2 H5 C2 H5 ¨0- CH3 ¨0- CH3 H C2H5 -OH -OH
O [OH
33 OCH3 OCH3 ¨0-8 cH3 OH

Date Regue/Date Received 2023-06-16 35a OCH3 OCH3 -(3-C CH3 H
-OH -OH

35c OCH3 OCH3 -(3-c cH3 -0-c cH3 benzyl H
-OH -OH

35d OCH3 OCH3 -o-c cH3 -o-c cH3 p-ropargyl H
OH OH

35e OCH3 OCH3 -(3-c cH3 -0-c cH3 allyl H
-OH -OH

36 H H -o-c cH3 -o-c cH3 OH OH

37 -0-c cH, -o-c cH3 OCH3 OCH3 CH3 H
-OH OH
Structures of Compound 21a-((1E,3Z,6E)-3-hydroxy-5-oxohepta- 1,3,6-triene-1,7-diy1)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2-hydroxymethyl)-2-methylpropanoate, Compound 35a-((1E,6E)-4,4-dimethyl-3,5-dioxohepta-1, 6-diene-1,7-diy1)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2-hydroxymethyl)-2-methylpropanoate, Compound 35d-((1E,6E)-3,5-dioxo-4,4-di(prop-2-yn-1-y1) hepta-1,6-diene-1,7-diy1)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2-(hydroxymethyl)-methylpropanoate), Compound 36-((1E,6E)-4,4-dimethy1-3,5- dioxohepta-1,6-diene-1,7-diy1)bis(4,1-phenylene)bis(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate), and Corn pound 37-((1E,6E)-4,4-dimethy1-3,5- dioxohepta-1,6-dien-1,7-diy1)bis(2-io methoxy-5,1-phenylene)bis(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate) are shown in Table 2.

Date Recue/Date Received 2023-06-16 Table 2 Compound Structure = OH
IVI ee0 " r, OM

21a HO 0== OH
OH
= 0 Me0 416 OlVie 35a HO"->ji0 411111)1P 0 OH
HO OH

I
35d HO"--)L0 36 He>fk0 0/11-eli HO OH

gir HO"->f0 OH
*INCOH IHO

The structure of Compound 21a includes a heptadiene-3,5-dione moiety, which can be readily interconvertible between the keto form and enol form. The 3- or OH group of the enol form combines with the adjacent 5- or 3-C=0 through hydrogen bonds to stabilize the structure thereof. In the present disclosure, two methyl functionalities are incorporated onto the 4-position of Compound 21a and afforded ((1E,6E)-4,4-dimethy1-3,5-dioxohepta-1,6-diene-1,7-diy1)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2-hydroxymethyl)-2-methylpropanoate (35a), which is found to possess stable keto form, and is not able to tautomerization. In addition, in Date Recue/Date Received 2023-06-16 the present disclosure, Compound 35a is used as a new lead compound and derived into a series of 4,4-dialkyl derivatives thereof (Compound 35a, Compound 35d, Compound 36 and Compound 37) with stable keto form.
2. Inhibitory growth effect of the diarylheptanoid compound of the present disclosure on the EGFR gene mutant NSCLC cells and the EGFR-TKIs resistant NSCLC cells The EGFR gene mutant NSCLC cells were treated with the diarylheptanoid compounds of the present disclosure, and then the cell survival was stained with crystal violet to measure the IC50 values of the diarylheptanoid compounds of the present disclosure in the EGFR gene mutant NSCLC cells, so as to determine the growth inhibitory effect of the diarylheptanoid compounds of the present disclosure on the EGFR gene mutant NSCLC cells.
EGFR gene mutations in lung cancer are mostly found in exons 18-21, which are the intracellular tyrosine kinase coding region. The most common mutations include E746-A750del in exon 19 and L858R point mutation in exon 21, which account for about 85%-90% of EGFR gene mutations. Tumor cells with these two mutations are sensitive to the EGFR-TKIs, known as activating mutations. Secondary mutations may occur in some tumor cells, and the most common secondary mutation is the T790M mutation in exon 20, which is a drug resistance mutation. Please refer to Fig. 1A, which shows the analysis result of the inhibition of the H1650 cells growth by the diarylheptanoid compounds of the present disclosure. The H1650 cells have the E746-A750del mutation in exon 19 of the EGFR gene, wherein the CCM represents curcumin and the BDMC represents bisdemethoxycurcumin. The IC50 value of the Date Recue/Date Received 2023-06-16 compound > 16 pM shows that the H1650 cells have not yet reached 50% cell growth inhibition after treatment with compounds at concentrations as high as 16 pM, and data in Fig. 1A are represented by mean SD (n = 3).
Fig. 1A shows the IC50 values in the H1650 cells treated with 22 diarylheptanoid compounds (including BDMC) and curcumin for 3 days. 18 diarylheptanoid compounds including Compound 1, Compound 24b, Compound 23b, Compound 33, Compound 25, Compound 35b, Compound 22a, Compound 21a, Compound 22b, Compound 26, Compound 36, Compound 35a, Compound 35c, Compound 27, Compound 31, Compound 37, Compound 35e and Compound 35d have significantly io better inhibitory activity than the parent compound - curcumin on the growth of the H1650 cells, wherein ** represents p <0.05, *** represents p <0.01, ****
represents p <0.001.
Experimentally, another 7 EGFR-TKIs resistant NSCLC cells were treated with Compound 21a, Compound 35a, Compound 35d, Compound 36 or Compound 37, and then the cell survival assay was performed to measure the IC50 values of the diarylheptanoid compounds of the present disclosure in the EGFR-TKIs resistant NSCLC cells, so as to determine the growth inhibitory effect of the diarylheptanoid compounds of the present disclosure on the EGFR-TKIs resistant NSCLC cells.
The EGFR-TKIs resistant NSCLC cells used in the test include the H1975 cells, the cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells, in which the H1975 cells have a point mutation of L858R in exon 21 and a secondary mutation of T790M in exon 20; the GR2 cells, the GR5 cells, the GR6 cells, the cells, the GR9 cells and the GR10 cells are gefitinib-resistant cell lines obtained after Date Recue/Date Received 2023-06-16 treating gefitinib with the HCC827 cells as parental cells, while the HCC827 cells and the H1650 cells are the NSCLC cells with E746-A750del mutation of EGFR exon 19.
Please refer to Fig. 1B, Fig. 1C and Fig. 1D. Fig. 1B shows the analysis result of the inhibition of the H1975 cells growth by the diarylheptanoid compounds of the present disclosure. Fig. 1C shows the analysis result of the inhibition of the cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells growth by the diarylheptanoid compounds of the present disclosure. Fig. 1D
shows the percentage changes of the IC50 values after the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells were treated with Compound 35d and gefitinib respectively. Data in Fig. 1B and Fig. 1C are presented as mean SD (n = 3), while the IC50 value > 16 pM show that the test cells have not yet reached 50% cell growth inhibition after treatment with compounds at concentrations as high as 16 pM. Gef in Fig. 1D represents gefitinib.
Fig. 1B shows the measured the IC50 values of the H1975 cells treated with Compound 21a, Compound 35a, Compound 35d, Compound 36, Compound 37 and curcumin for 3 days, respectively. The results showed that Compound 21a, Compound 35a, Compound 35d, Compound 36 and Compound 37 of the present disclosure had significantly better inhibitory activity than curcumin on the growth of the H1975 cells.
Fig. 1C shows the measured the IC50 values of the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells after treatment with Compound 21a, Compound 35a, Compound 35d, Compound 36, Compound 37 and curcumin for 3 days, respectively.
The results show that Compound 21a, Date Recue/Date Received 2023-06-16 Compound 35a, Compound 35d, Compound 36 and Compound 37 of the present disclosure have significantly better inhibitory activity than curcumin on the growth of the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells.
The results in Fig. 1B and Fig. 1C show that all EGFR-TKIs resistant NSCLC
cells are more sensitive to Compound 21a, Compound 35a, Compound 35d, Compound 36 and Compound 37 than curcumin, and can achieve 50% inhibition of cell growth at significantly lower concentrations.
Fig. 1D shows that the analysis results obtained by comparing the IC50 values io measured by the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells with the IC50 value measured by the HCC827 cells, in which the HCC827 cells, the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells were treated with Compound 35d and gefitinib for days and then measured the IC50 values. The results in Fig. 1D show that all is gefitinib-resistant cell lines are indirectly sensitive to Compound 35d.
All gefitinib-resistant cell lines were >200-fold more resistant to gefitinib (Gef) than their parental cells, the HCC827 cells.
3. Anticancer activity of Compound 35d against the GR6 tumor, the GR8 tumor and the HCC827 tumor 20 In order to verify the anticancer effect of the diarylheptanoid compound of the present disclosure in vivo, the GR6 tumor mouse model, the GR8 tumor mouse model and the HCC827 tumor mouse model of xenotransplantation were first established, and the GR6 tumor mice, the GR8 tumor mice, and the HCC827 tumor mice were Date Regue/Date Received 2023-06-16 treated with 100 mg/kg of Compound 35d daily for 35 days, and the tumor size and body weight of the GR6 tumor mice, the GR8 tumor mice, and the HCC827 tumor mice were recorded.
Please refer to Fig. 2A to Fig. 2D, Fig. 2A shows the analysis result of the inhibition of tumor growth in the GR6 tumor mice by treating Compound 35d alone, Fig. 2B shows the analysis result of the inhibition of tumor growth in the GR8 tumor mice by treating Compound 35d alone, Fig. 2C shows the analysis result of the inhibition of tumor growth in the HCC827 tumor mice by treating Compound 35d alone, and Fig. 2D shows the statistical chart of body weight changes in the tumor mice by io treating Compound 35d alone, wherein data in Fig. 2A to Fig. 2C are presented as mean SEM (n = 10).
The results in Fig. 2A to Fig. 2C show that Compound 35d significantly inhibits the tumor growth of the GR6 tumor mice and the GR8 tumor mice, but has less significant effect on inhibiting the tumor growth of the HCC827 tumor mice.
However, the results in Fig. 2D show that the body weight of the tumor mice did not decrease significantly after being treated with Compound 35d for more than 1 month.
4. Combined treatment of Compound 35d and osimertinib inhibits the re-progression of the GR6 tumor EGFR-TKIs are currently the standard treatment for NSCLC patients with the EGFR gene mutation. To further test whether the combined use of the diarylheptanoid compound of the present disclosure and known ones can enhance the therapeutic effect of NSCLC, the GR6 tumor mice were divided into 4 groups.
One group received 100 mg/kg Compound 35d treatment per day (represented as Date Recue/Date Received 2023-06-16 35d), another group received 1 mg/kg osimertinib treatment per day (represented as Osi), still another group received the combined treatment of 100 mg/kg Compound 35d and 1 mg/kg osimertinib per day (represented as 35d+Osi), and the other group was the control group without drug treatment.
Please refer to Fig. 3A and Fig. 3B, Fig. 3A shows the analysis result of the inhibition of tumor re-progression in the GR6 tumor mice by treating the combined treatment of Compound 35d and osimertinib, and Fig. 3B shows the statistical chart of body weight changes in the GR6 tumor mice by treating the combined treatment of Compound 35d and osimertinib, wherein data in Fig. 3A and Fig. 3B are .. represented by mean SEM (n = 10).
The results in Fig. 3A show that although the tumor size of the GR6 tumor mice in the Osi group was initially reduced by osimertinib treatment, the treated tumors subsequently re-growth, indicating that the tumors of the GR6 tumor mice have EGFR-TKIs resistance. However, no matter in the group treated with Compound 35d alone or in the group treated with the combined treatment of Compound 35d and osimertinib, tumor re-progression in the GR6 tumor mice was significantly inhibited.
And the results in Fig. 3B show that neither the single treatment of Compound 35d nor the combined treatment of Compound 35d and osimertinib can significantly reduce the body weight of mice.
To sum up, the present disclosure provides a new use of the pharmaceutical composition, which can be used to manufacture a drug for treating lung cancer.
The pharmaceutical composition includes the diarylheptanoid compound or the pharmaceutically acceptable salt thereof, which can inhibit the growth of the EGFR

Date Recue/Date Received 2023-06-16 gene mutant NSCLC cells and the EGFR-TKIs resistant NSCLC cells, so that can be used to manufacture the drug for treating lung cancer.
Moreover, the pharmaceutical composition can have a synergistic effect when used in combination with EGFR-TKIs, which can increase the effectiveness of treating lung cancer, especially for the treatment of lung cancer with the EGFR gene mutation and EGFR-TKIs, and has the potential to be used in the medical and health care market.
Although the present disclosure has been described as above by way of embodiments, it is not intended to limit the present disclosure. Person having skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure should be defined by the scope of the appended patent application.

Date Recue/Date Received 2023-06-16

Claims

WHAT IS CLAIMED IS:

1. A use of a pharmaceutical composition, characterized in that, the pharmaceutical composition is used to manufacture a drug for treating a lung cancer, wherein the pharmaceutical composition comprises a diarylheptanoid compound or a pharmaceutically acceptable salt thereof, the diarylheptanoid compound has a structure represented by Formula (l):

Ra R
a Rb RC RC
Rbi Re Re Formula (l), wherein Ra, Rb, Ra' and Rb' are independently H, C1-C2 alkyl, C1-C3 alkoxy, OH, or -0C(=0)Rd, wherein Rd is C1-C3 alkyl or Cl-C3 alkanol; Rc is H, C1-C2 alkyl, C3-C6 unsaturated alkyl or C7-C12 arylalkyl with double or triple bonds; and Re and Re' are independently H, C1-C6 alkyl or C1-C6 alkoxy.

2. The use of the pharmaceutical composition of claim 1, wherein at least one of Ra, Rh, Ra', and Rb' is -0C(=0)Rd, wherein Rd is C1-C3 alkyl or C1-C3 alkanol.

3. The use of the pharmaceutical composition of claim 1, wherein the diarylheptanoid compound is interconvertible between keto form and enol form, when Rc is H.

Date Recue/Date Received 2023-06-16

4. The use of the pharmaceutical composition of claim 1, wherein the diarylheptanoid compound is selected from Compound 1, Compound 21a, Compound 21b, Compound 22a, Compound 22b, Compound 23a, Compound 23b, Compound 24a, Compound 24b, Compound 25, Compound 26, Compound 27, Compound 31 and Compound 33 having a structure represented by Formula (II):

R1 --" R1' Formula (II);
wherein Ri, Ri' are OCH3 respectively, R2, R2' are H respectively, R3, R3' are H respectively in Compound 1; Ri, Ri' are OCH3 respectively, R2, R2' are 0R4 respectively, R3, R3' are H respectively in Compound 21a; Ri, Ri' are OCH3 respectively, R2 iS OH, R2' iS 0R4, R3, R3' are H respectively in Compound 21b; Ri, Ri' are OCH3 respectively, R2, R2' are 0R5 respectively, R3, R3' are H
respectively in Compound 22a; Ri, Ri' are OCH3 respectively, R2 iS OH, R2' is OR5, R3, R3' are H
respectively in Compound 22b; Ri, Ri' are H respectively, R2, R2' are ORs respectively, R3, R3' are H respectively in Compound 23a; Ri, Ri' are H
respectively, R2 iS OR5, R2' iS OH, R3, R3' are H respectively in Compound 23b; Ri, Ri' are respectively, R2, R2' are OCH3 respectively, R3, R3' are H respectively in Compound 24a; Ri is 0R4, Ri' is OH, R2, R2' are OCH3 respectively, R3, R3' are H
respectively in Compound 24b; Ri is ORs, Ri' is OH, R2, R2' are OCH3 respectively, R3, R3' are H

Date Recue/Date Received 2023-06-16 respectively in Compound 25; Ri, Ri' are 0C2H5 respectively, R2, R2' are 0R4 respectively, R3, R3' are H respectively in Compound 26; Ri, Ri' are 0C2H5 respectively, R2, R2' are OR5 respectively, R3, R3' are H respectively in Compound 27;
Ri, Ri' are C2H5 respectively, R2, R2' are OR5 respectively, R3, R3' are C2H5 respectively in Compound 31; and Ri, Ri' are OCH3 respectively, R2 is OCH3, R2' iS
0R4, R3, R3' are H respectively in Compound 33;
wherein R4 is a structure represented by Formula (i), and R5 is a structure represented by Formula (ii):

ÇOH
VrOH
OH OH
Formula (i); Formula (ii).

5. The use of the pharmaceutical composition of claim 1, wherein the diarylheptanoid compound is selected from Compound 35a, Compound 35c, Compound 35d, Compound 35e, Compound 36 and Compound 37 having a structure represented by Formula (III):

R6 ss.. R6' ix7 Formula (III);
Date Recue/Date Received 2023-06-16 wherein R6, R6' are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is CH3 in Compound 35a; R6, Re are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is benzyl in Compound 35c; R6, Re are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is propargyl in Compound 35d; R6, Re are OCH3 respectively, R7, R7' are 0R4 respectively, R8 is allyl in Compound 35e; R6, Re are H respectively, R7, R7' are 0R4 respectively, R8 is CH3 in Compound 36; and R6, Re are 0R4 respectively, R7, R7' are OCH3 respectively, R8 is CH3 in Compound 37;
wherein R4 is a structure represented by Formula (i):
ÇOH
Formula (i).

6.
The use of the pharmaceutical composition of claim 1, wherein the diarylheptanoid compound is selected from:
meoNja = 0 OH HO'>
HO
**"011.1 HO

= =
Me 0Me 0 0, ?CQH
I I
HO OH
and o 0 He>fil*'0 OH

HsCO OCH3 Date Recue/Date Received 2023-06-16

7. The use of the pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKls).

8. The use of the pharmaceutical composition of claim 7, wherein the EGFR-TKls are osimertinib, gefitinib, erlotinib or afatinib.

9. The use of the pharmaceutical composition of claim 1, wherein the lung cancer is a non-small cell lung cancer.

10. The use of the pharmaceutical composition of claim 1, wherein the lung cancer is resistant to epidermal growth factor receptor-tyrosine kinase inhibitors.

Date Regue/Date Received 2023-06-16